Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections

Significance BET bromodomain inhibition is therapeutic in multiple diseases; however, pan-BET inhibitors have induced significant myelosuppression and gastrointestinal toxicity, perhaps due to inhibition of both tandem bromodomains (BD) of all BET family members. However, selective inhibition of just the first BD (BD1) phenocopies pan-BET inhibitor activity in preclinical models of cancer, other diseases, and, for BRDT, in the testes for a contraceptive effect. Here, we leveraged our multibillion-molecule collection of DNA-encoded chemical libraries (DECLs) to identify BET BD1-selective inhibitors of specific chirality with high potency, stability, and good cellular activity. Our findings highlight the robustness and efficiency of the DECL platform to identify specific, potent protein binders that have promise as potential anticancer and anti-inflammatory agents and as male contraceptives.


Syntheses of Compounds
1a. Materials and instrumentation. All reactions involving air-sensitive reagents were carried out in anhydrous solvents under an atmosphere of nitrogen. Reagents and solvents purchased from commercial supplies were used as received. Reactions were monitored by thin-layer chromatography (TLC) on Bakerflex ® silica gel plates (IB2-F) using UV-light (254 and 365 nm) detection or high-performance liquid chromatography/mass spectrometry (HPLC-MS). Column chromatography was carried out using Teledyne ISCO CombiFlash system equipped with either a silica or C-18 column. NMR spectra were recorded at room temperature using a Bruker Avance III HD 600 MHz spectrometer ( 1 H NMR at 600 MHz and 13 C NMR at 150 MHz) or a Bruker Avance III HD 800 MHz spectrometer ( 13 C NMR at 200 MHz). Chemical shifts (δ) are reported in parts per million (ppm) with reference to solvent signals [ 1 H-NMR: DMSO-d6 (2.50 ppm); 13 C-NMR: ]. Signal patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), h (heptet), m (multiplet) and br (broad). Coupling constants (J) are given in Hz. HRMS measurements were performed using ThermoFisher Scientific Q Exactive instrument. Abbreviations presented in experimental procedures are referred to the following definitions: DIEA, N,N-diisopropylethylamine; DME, 1,DMF,N,EtOAc,ethyl acetate;HATU,N,N',NaOH(aq), aqueous sodium hydroxide solution; Na2CO3(sat), saturated aqueous sodium carbonate solution; Na2SO4, sodium sulfate.
The compounds (2a-2c) were prepared in a way similar to the procedure described above.

S5
The compounds (4a-4d) were prepared in a way similar to the procedure described above.     Compound 4a (322 mg, 0.84 mmol, 1 eq) was dissolved in DMF (3 ml) containing piperidine (10% v/v) and stirred 1h in room temperature. Resulting mixture is evaporated to The SM was dissolved in DMF containing piperidine, and all solvents were removed after 20 min. After completing reaction all solvents were removed in vacuo and kept overnight dry in high vacuo. To the same vessel mixture of DIEA (439 μL, 2.5 mmol, 3 eq), 5-(2,4-dimethylphenyl) picolinic acid (5a) (140 mg, 0.7 mmol, 0.8 eq) and HATU (957.6 mg, 2.5 mmol, 3 equiv.) added in the solution of anhydrous DMF (8 ml) under nitrogen. The aqueous layer was extracted twice with EtOAc, and the combined organic extracts were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (CH3OH/CH2Cl2, 1:99 to 5:95) to afford 4a (327 mg, 68%) as a white solid.
The compounds (11a-11b) were prepared in a way similar to the procedure described above.
The compounds (12a-12g) were prepared in a way similar to the procedure described above.

Mosher Method.
We utilized the Mosher method to determine the absolute stereochemistry of α-chiral carbon of piperidine propanoic acid 3R. Two possible isomers R and S are possible at the α-carbon; to identify this, we have synthesized MTPA adducts 4 (R, R) (R-MTPA amide) and 4 (R, S) (S-MTPA amide) and carried out chiral analysis by determining ΔδSR (ppm). Detailed synthetic steps depicted in Scheme 3. Substituted piperidine propanoic acids (1R) undergo sequential addition of O- (7-azabenzotriazol-1-yl Table 4. Protons on position 3 and 4, adjacent to the α-carbon have shown positive values of ΔδSR (ppm) and ΔδSR (Hz), which indicates below the plane. Whereas protons on position 10 shows above the plane and shown negative values of ΔδSR (ppm) and ΔδSR (Hz). Detailed H1 NMR spectra of MTPA amides are mentioned in supporting data. From this experiment it is clear that compound 3R is R isomer and its corresponding most active compounds CDD-787 and CDD-956 were also R isomers, which are also in agreement with our positive optical rotation.
Scheme S2. Scheme for the synthesis of MTPA amide from 1R. Reagents and conditions: i) Methylamine HCl, O- N,N',

(R, R)
R-MTPA amide   were produced as previously described (1)(1)(1)(1)(1)(1). In brief, cDNA encoding human BRDT and BRD4 BD1 and BD2 bromodomains were subcloned into pET15b or pET28b bacterial vectors (Addgene, USA) with an N-terminal polyhistidine tag (6-His; linear) for expression and purification. His-tagged recombinant protein was isolated and eluted by immobilized metal affinity chromatograph followed by size exclusion gel filtration chromatography. Each BD was confirmed to be properly folded and active by a fluorescence thermal shift stability assay and AlphaScreen with biotinylated JQ1 as ligand, respectively.

DEC-Tec affinity selection with bromodomain proteins.
To identify BRDT-BD1 selective compounds, we screened our DEC-Tec library pool. We had five screening conditions: 1) absence of bromodomain proteins (bead binding non-target control, NTC); 2) presence of His-BRDT-BD1 at 0.3 μM; 3) presence of His-BRDT-BD2 at 0.3 μM (a counter-screen for bromodomain selective compounds); 4) presence of His-BRDT-BD1 plus JQ1 at 100 μM; and 5) presence of His-BRDT-BD2 plus JQ1 at 100 μM. After three rounds of DEC-Tec selection, the DNA barcode from the last round of selection was PCR amplified.
Following cleanup by Agencourt AMPure XP beads and quantitation with Agilent high sensitivity DNA kit using a Bioanalyzer, the DNA was sequenced in a single-read 105-cycle sequencing on an Illumina NextSeq 500 instrument.
Bromodomain proximity assay. The AlphaScreenTM assay was performed following previous publication with minor modifications from the manufacturer's protocol (PerkinElmer, USA). A 20-μL reaction was set up in a PerkinElmer 384-well AlphaPlate where His-bromodomain at 10 nM was S46 incubated with biotinylated JQ1 at 10 nM, nickel chelate acceptor beads at 12.5 μg/mL, and tested compound at various concentrations for 15 min at room temperature, followed by the addition of streptavidin donor beads at 12.5 μg/mL and another 60-min incubation at room temperature. The plate was read on a Tecan Infinite M1000 Pro plate reader.
Thermal shift assay. The dye SYPRO Orange (ThermoFisher Scientific, USA) was used to perform the protein thermal shift assay. The assay was set up on a 384-well Roche plate where His-bromodomain at a concentration of 2 μM was incubated with the test compound at various concentrations, and SYPRO Orange dye at 5 × in a 10-μL reaction. The melting curve experiment and data analysis was run on a Roche Lightcycler 480 real-time PCR instrument. BromoKdELECT assays. Assays were performed at Eurofins DiscoverX (Fremont, CA) using the following procedures. T7 phage strains displaying bromodomains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection = 0.4) and incubated with shaking at 32°C until lysis (90-150 minutes). The lysates were centrifuged (5,000 x g) and filtered (0.2μm) to remove cell debris.

Metabolic stability assay in liver microsomes
Streptavidin coated magnetic beads were treated with biotinylated small molecule or acetylated peptide